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J Physiol (2003), 550.3, pp. 819-828
© Copyright 2003 The Physiological Society
DOI: 10.1113/jphysiol.2003.041970
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ABSTRACT |
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Photorelaxation of vascular smooth muscle (VSM) was studied using segments of tail artery from normotensive rats (NTR) or spontaneously hypertensive rats (SHR). Isolated vessels with intact endothelium were perfused with Krebs solution containing phenylephrine. Perfusion pressures were recorded while arteries were irradiated with either visible (VIS;= 514.5 nm) or long wavelength ultra-violet (UVA;
(Received 20 February 2003; accepted after revision 12 May 2003; first published online 24 June 2003)
Corresponding author F. W. Flitney: Cell and Molecular Biology, School of Biology, University of St Andrews, St Andrews, Fife KY16 9TS, Scotland. Email: fwf{at}st-and.ac.uk
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INTRODUCTION |
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Vascular smooth muscle (VSM) relaxes when irradiated with visible or ultra-violet (UV) light (Furchgott et al. 1961). The discovery of a potent relaxing factor released from endothelial cells (Furchgott & Zawadski, 1980), later identified as nitric oxide (NO; Palmer et al. 1987), led to the suggestion that photorelaxation might be caused by a light-activated relaxing factor with pharmacological properties similar to NO (Matsunaga & Furchgott, 1989; Furchgott & Jothianandan, 1991). This hypothesis rested on certain similarities between endothelium-dependent relaxation and photorelaxation: both types of response are accompanied by elevated intracellular cyclic GMP levels and are blocked by oxyhaemoglobin, a NO scavenger, or by methylene blue, an inhibitor of soluble guanylate cyclase (Karlsson et al. 1984; Martin et al. 1985).
The idea that NO might mediate photorelaxation has been substantiated by recent studies. Megson et al. (1995) showed that irradiating a perfused, intact artery with monochromatic ( = 514.5 nm) light produced a transient vasodilator response that reversed fully during the period of illumination (6 min). The vessel was unable to respond to a second, identical period of irradiation delivered immediately after the first, but its photosensitivity returned slowly if it was allowed to remain in the dark. The recovery process, called repriming, displayed an absolute requirement for endothelium-derived NO: it could be prevented by NG-monomethyl-L-arginine (L-NMMA), an NO synthase (NOS) inhibitor; by adding oxyhaemoglobin to the perfusate; or by physically removing the endothelium. Repriming could also be blocked by ethacrynic acid, a thiol-alkylating agent.
Based upon these observations, it was postulated that VSM contains a photolabile 'store' of NO that can be discharged rapidly by exposing a vessel to visible light (Megson et al. 1995). The inability to produce a second response immediately afterwards shows that the capacity of the store is both finite and exhaustible and an obligatory period in the dark is required afterwards to reconstitute it. Since the repriming process requires endogenous (endothelial) NO and tissue thiols, it seemed likely that the photosensitive material could be a nitrosothiol (RSNO). Glutathione (GSH) is the most abundant tissue thiol and its S-nitroso adduct, S-nitrosoglutathione (GSNO), releases NO when exposed to visible or UV light (Sexton et al. 1994; Singh et al. 1996; Wood et al. 1996). A study showing that repriming is attenuated in vessels deficient in GSH but enhanced in those with elevated GSH levels (Megson et al. 2000) provided support for the idea that GSNO could be a component of the photosensitive 'store'. Recent measurements of the action spectrum for photorelaxation show a peak response in the region of 330-340 nm (Rodriguez et al. 2003), consistent with this conclusion.
The 'molecular store' model outlined above assigns an essential role for the endothelium in photorelaxation and offers a rational explanation for the manner in which a functionally intact artery responds to visible light. However, it is well-known that endothelium-denuded vessels relax when exposed to light (Matsunaga & Furchgott, 1989; Chen & Gillis, 1992, 1993; Venturini et al. 1993; Kubaszewski et al. 1994; Charpie et al. 1994; Goud et al. 1996) and they are able to do so repeatedly, even though there is no source of endothelial NO available between successive periods of illumination. This seems at odds with the idea that photorelaxation is dependent upon endothelium-derived NO. However, there are a number of factors that might contribute to this discrepancy. First, most studies have used endothelium-denuded vessels, irradiated with either UV light (Matsunaga & Furchgott, 1989; Chen & Gillis, 1992, 1993; Kubaszewski et al. 1994; Charpie et al. 1994; Goud et al. 1996) or with polychromatic light containing a significant UV component (Venturini et al. 1993). Few studies have been conducted using preparations with an intact endothelium (Furchgott et al. 1961; Lovren et al. 1996; Lovren & Triggle, 1998; Rodriguez et al. 2003) and fewer still using intact, perfused vessels exposed to monochromatic visible light (Megson et al. 1995; 2000). Second, several studies have shown that some NOS inhibitors enhance photorelaxation (Chen & Gillis, 1992; Charpie et al. 1994) and these cast further doubt on the importance of the endothelium. However, some of the inhibitors used (e.g. L-NAME) will generate NO if exposed to light (Bauer & Fung, 1993; Chang et al. 1993; Lovren et al. 1996) and this probably explains why they potentiate photorelaxation. Finally, photorelaxation is reportedly enhanced in vessels from hypertensive animals (Charpie et al. 1994; Kubaszewski et al. 1994). This also argues against a role for the endothelium, since vessels from hypertensive animals generally show blunted endothelium-dependent relaxations (Winquist et al. 1984; Bauersachs et al. 1998; Vallance, 1999), as do those of patients with essential or secondary hypertension (Puddi et al. 2000; Lind et al. 2000).
The purpose of the present study was to try and reconcile some of these conflicting observations. Accordingly, responses to both visible and UV light were studied. Experiments were made with intact rat tail artery segments perfused at a constant flow rate and responses obtained with vessels from normotensive rats were compared to those from age-matched spontaneously hypertensive animals.
The results of this study were presented at a NATO-ASI meeting on vascular endothelium (Flitney & Megson, 2001).
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METHODS |
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All experimental procedures used in this study conformed to the UK Animals (Scientific Procedures) Act 1986.
Preparation
Experiments were performed on functionally intact, isolated segments of pre-contracted tail arteries (1-2 cm) from age-matched normotensive rats (NTR) or spontaneously hypertensive rats (SHR) (Charles River, Margate, UK). Animals were killed by cervical dislocation. The tail artery was dissected free and attached to a plastic cannula using a fine thread. The length and external diameter of each segment was measured using a travelling microscope fitted with an eyepiece graticule. The cannulated vessel was then connected to a perfusion system driven by a peristaltic pump (Flitney et al. 1992). The apparatus was modified for the purpose of this study to permit four arteries (2 SHR and 2 NTR) to be perfused simultaneously.
Light sources
Arteries were illuminated using one of two sources: an argon ion laser (Spectra Physics Ltd, type 186-09), tuned to operate at a wavelength of 514.5 nm (VIS light), or a UV source (Mineralite lamp, type UVGL-58; UVP Inc., San Gabriel, CA, USA) that emitted at 366 nm (UVA light). Light from the laser was directed onto the preparation via a fibre optic bundle. Vessels were illuminated with UVA light by positioning the lamp 1 cm above the perfusion chamber. Output intensities were measured using a Spectra Physics power meter (Type 404). Irradiances at the preparation were estimated to be 6.3 mW cm-2 for 514.5 nm and 1.2 mW cm-2 for 366 nm).
Experimental protocols
All experiments were conducted under subdued laboratory lighting (60 W red safelight, ~2 m from the perfusion apparatus). Arteries were perfused with Krebs solution (composition (mM): NaCl, 118; KCl, 4.7; NaHCO3, 25; NaH2PO4, 1.15; CaCl2, 2.5; MgCl2, 1.1; glucose, 5.6; gassed with 95 %O2-5 %CO2 to maintain a pH of 7.4; temperature 31-33 °C), initially at a flow rate of 0.2 ml min-1. The flow was increased stepwise over the next 20 min to 2 ml min-1 and preparations were then allowed to stabilise for a further 10-15 min before adding a standard concentration of 1 µM phenylephrine (PE) to the perfusate. Perfusion pressures were recorded before (passive) and after (active = total - passive) the addition of 1 µM PE. The [PE] was then adjusted incrementally until stable working pressures of ~130 mmHg were obtained.
Repriming curves were constructed by first exposing vessels to a 'conditioning' period (6 min) of VIS or UVA illumination at the irradiances specified above. They were then subjected to a series of identical periods of irradiation, after spending different intervals (
T = 10, 20, 40, 72, 150 and 300 min) recovering in the dark. The amplitude of each photorelaxation was measured (P/P 
102, where P is the perfusion pressure immediately before irradiating the vessel, and P is the maximum pressure drop) and plotted as a function of T.
Repriming curves were fitted to a hyperbolic function (eqn (1)) using GraphPad Prism software (Megson et al. 2000), employing an iterative procedure that was terminated when the sum of squares changed by < 0.01 %:
y = (bx)/(c + x), (1)
where y = P/P, b = P/Pmax, c = T1/2, the time at which P/P = 1/2(P/Pmax) and x = T.
Cardiovascular parameters of NTR and SHR
Measurements of blood pressure and heart rate were made on two groups (n = 5) of age-matched NTR and SHR using a tail plethysmograph (type 179 Blood Pressure Analyser; ITIC Life Science, USA). Recordings were made on conscious rats, placed on a warmed (25-27 °C) stage and restrained in a Perspex tube. Pressures were recorded daily for 4 weeks to familiarise the animals to the procedure and to reduce stress. Mean (± S.E.M.) values for blood pressures and heart rate were calculated from measurements made during the four days immediately before killing.
Compounds and sources
L-phenylephrine-HCl and bovine haemoglobin were obtained from Sigma Ltd. Methaemoglobin was reduced to haemoglobin by reaction with sodium dithionite, as previously described (Megson et al. 1995). L-NMMA was a generous gift from Dr Harold Hodson, Wellcome Research Laboratories, Kent. Other reagents were Analar grade obtained from BDH Ltd.
Statistical analyses
The statistical significance of differences between repriming curves was assessed by two-way ANOVA. Student's t test was used as appropriate to establish differences between paired and unpaired data. Results were considered statistically significant for P < 0.05.
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RESULTS |
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Blood pressure measurements and physiological parameters of arteries from NTR and SHR
The results are summarised in Table 1. Blood pressure measurements confirmed that age-matched SHR had significantly higher systolic (P < 0.001) and diastolic (P < 0.001) pressures than NTR. The heart rates of the two groups of animals were not significantly different.
Arteries from SHR that were perfused with Krebs solution alone exhibited a higher passive resistance to flow than those isolated from NTR: the mean perfusion pressure was ~1.5 times greater (P < 0.05). Vessels from SHR had significantly smaller external diameters (P < 0.0001) that might account for their increased resistance to flow. The [PE] required to generate the same working perfusion pressure (~130 mmHg) was ~1.35 times greater for vessels from NTR than those from SHR (P < 0.05).
Characteristics of VIS and UVA light-induced photorelaxations
VIS and UVA light-induced photorelaxations are qualitatively different. Arteries differed in their response to VIS and UVA light. Figure 1 shows averaged recordings (n = 7) for both types of response. The recordings were made using vessels from NTR after allowing them to recover for 72 min in the dark following an initial conditioning response. VIS light-induced photorelaxations were transient in nature, comprising a rapid decrease of pressure that reversed during the period of illumination (Fig. 1A). In contrast, the response of a vessel to UVA light comprised an initial rapid vasodilatation, followed by a partial recovery only (Fig. 1B). The residual level of reduced tone was maintained until the light was switched off, at which time the perfusion pressure quickly returned to the level that existed before illuminating the vessel.
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Figure 1. UVA light-induced photorelaxations comprise two vasodilator components Time-averaged recordings of intact tail arteries (n = 7) irradiated with visible or UVA light. A, visible light produces a monophasic vasodilator response that reverses during the period of illumination. B, the response to UVA light is a composite one and results from the summation of two identifiable elements: a phasic component (p) that resembles the response to visible light (C), superimposed on a sustained (s) component that persists throughout the period of irradiation (D). Scale bar: 10 % for visible and 5 % for UVA light. Period of illumination (6 min) indicated by the step-up in the top traces. | ||
The form of the UVA light-induced response suggests that it is the sum of two vasodilator elements: a brief phasic component, superimposed upon a sustained component that persists for as long as the vessel remains illuminated. In what follows, we will refer to these as the p- and s-components of the UVA light-induced response, respectively (Fig. 1D).
VIS light-induced responses and the p-component of UVA light-induced photorelaxations are both due to the release of NO from a photosensitive molecular 'store'. Irradiating a vessel with VIS light produces a transient photorelaxation, as noted above. The same artery is incapable of responding to a second, identical period of illumination if this is delivered soon (< 1-2 min) after the first, but its photosensitivity recovers in the dark (Fig. 2). The response of an artery to repeated periods of illumination using the UVA source is different. A vessel previously exposed to UVA light will respond to a further period of irradiation even when given immediately after the first, but the second response invariably lacks a discernible phasic element. However, if the vessel is allowed to remain in the dark then this component returns gradually. This is illustrated in Fig. 3. The filled circles are original recordings that were made at the times indicated. The open circles show the form of the p-component only, obtained by subtracting the second response (in this case, recorded 10 min after the first) from the third (40 min) and subsequent (72, 150 and 300 min) responses. Repriming of the p-component is qualitatively similar to that seen when vessels are irradiated with VIS light, since it too can be prevented by treating a vessel with either L-NMMA (Fig. 4) or haemoglobin (Hb, 5 µM; not shown).
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Figure 2. Repriming of a vessel exposed to VIS light Pressure recordings showing the time course of the recovery of an artery following exposure to visible light. The first in a series of photorelaxations (0 min) depletes the store so that the vessel cannot respond immediately to a second period of illumination. The ability to do so returns slowly when the artery is allowed to remain in the dark. Scale bar = 10 %. | ||
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Figure 3. Repriming of the p-component of UVA light-induced responses Time-averaged pressure recordings (n = 5) showing repriming of the phasic (p-) component ( | ||
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Figure 4. Effect of L-NMMA on repriming of UVA light-induced photorelaxations Repriming of the p-component of the UVA-induced response (shown in Fig. 3) is blocked in the presence of L-NMMA but the s-component is not. | ||
The s-component of the UVA response is independent of endothelial NO. The s-component of the UVA light-induced response is not affected by L-NMMA (Fig. 4) or Hb (not illustrated). It can therefore be studied in isolation by treating a vessel with L-NMMA to prevent repriming of the p-component. Under these conditions, the amplitude of each response is virtually independent of the time interval (T) between successive periods of illumination (Fig. 4). Thus, the underlying mechanism does not depend directly upon endothelial NO and is therefore quite distinct from both the p-component and the VIS light-induced response.
These observations suggest that UVA and VIS light can both liberate NO from a molecular store, but that only the more energetic UVA light can activate the process(es) responsible for the s-component. This is supported by the experiment shown in Fig. 5. Here, prior exposure to UVA light abrogated the response to VIS light (Fig. 5B) but when the illumination sequence was reversed, the p-component of the UVA response was eliminated while the s-component remained (Fig. 5A).
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Figure 5. VIS and UVA light can induce the phasic response, but only UVA light can elicit the sustained response A, irradiating a vessel with VIS light produces a monophasic photorelaxation. When the same vessel is exposed to UVA light immediately afterwards the p-component is absent but the s-component remains. B, the reverse pattern of illumination eliminates the response to VIS light altogether. | ||
Quantitative analysis of repriming curves
Repriming of VIS light-induced photorelaxation is impaired in vessels from SHR. Figure 6 shows repriming curves for VIS light-induced responses obtained with vessels from NTR and SHR. The results obtained with vessels from NTR (Fig. 6A) are in good agreement with those published previously (Megson et al. 1995, 2000): they show P/P approaching an asymptote (P/Pmax) of 51.1 ± 1.8 %, with a T1/2 of 134 ± 10 min. This is in marked contrast to what is seen using vessels from SHR (Fig. 6B). Here the repriming process is significantly impaired: P/Pmax is only 25.1 ± 1.9 % with a T1/2 of 54.0 ± 12 min. A two-way ANOVA showed that this difference is significant at the P < 0.05 level.
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Figure 6. Repriming curves for arteries from normotensive rats Repriming curves obtained by irradiating arteries from normotensive rats with either VIS (A) or UVA (B) light. The amplitudes of VIS light-induced photorelaxations are considerably greater than for UVA light-induced responses and the repriming process takes longer to complete. | ||
Repriming of the p-component of UVA light-induced photorelaxations. Figure 7 shows repriming curves for UVA light-induced photorelaxations. In Fig. 7A and B the initial (maximum) decrease of pressure is plotted (upper curves) together with the amplitude of the s-component (s in Fig. 1D; lower curves) for arteries from both NTR (Fig. 7A) and SHR (Fig. 7B). The data show that repriming is attenuated in vessels from NTR as compared to SHR. P/Pmax is 16.2 ± 0.4 % with a T1/2 of 16.5 ± 1.8 min for NTR arteries (Fig. 7A), as compared to 28.5 ± 0.93 % and 28.6 ± 3.2 min, respectively, for vessels from SHR (Fig. 7B).
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Figure 7. Repriming of vessels from SHR and NTR irradiated with UVA light Repriming curves obtained by irradiating vessels from NTR (A) and SHR (B) with UVA light. Upper curves in each panel show peak (initial) pressure drop (see Fig. 1B) and lower curves show amplitude of s-component. C and D show repriming curves for the p-component only for vessels from NTR and SHR, respectively. Details of the method used to analyse the curves are given in the text. | ||
The amplitude of the p-component alone can be estimated by subtracting the residual vasodilatation, measured immediately before extinguishing the light, from the initial rapid fall of pressure (see diagram of Fig. 1). Repriming curves representing the time course for recovery of the p-component alone are shown in Fig. 7C and D. Analysis of these curves give values for P/Pmax and T1/2 of 12.1 ± 0.4 % and 19.7 ± 2.3 min, respectively, for vessels from NTR (Fig. 7C). The corresponding values for arteries from SHR are 23.1 ± 1.71 % and 61.2 ± 12.4 min (Fig. 7D). Thus, repriming of photorelaxation caused by UVA irradiation is enhanced in vessels from SHR as compared to arteries from NTR. This is the reverse of what was seen when arteries from SHR and NTR were illuminated with VIS light (Fig. 5).
Repriming of VIS light-induced and the p-component of UVA light-induced photorelaxations are virtually identical for vessels from SHR. The above analysis shows that the repriming curve for VIS light-induced photorelaxations and the corresponding curve for the p-component of UVA light-induced responses are strikingly similar when studying vessels from SHR. This is illustrated in Fig. 8 where both data sets (from Fig. 6B and Fig. 7D) are plotted together. Statistical analysis (two-way ANOVA) shows that the two curves are not significantly different (P > 0.05).
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Figure 8. Repriming curves for VIS light-induced and p-component of UVA light-induced responses are virtually identical for vessels from SHR Repriming of the p-component of vessels from spontaneously hypertensive rats exposed to UVA light ( | ||
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DISCUSSION |
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Qualitative considerations
This study reveals qualitative differences between photorelaxations generated by VIS light and those evoked by UVA light. Our experiments show that UVA light-induced photorelaxations are due to the superimposition of two vasodilator components that possess distinctly different properties (Flitney & Megson, 2001). The p-component is indistinguishable from the VIS light-induced response: both are transient in nature, caused by the rapid discharge of NO from a molecular 'store' that can be regenerated afterwards in the dark. Repriming of these types of response can be prevented by perfusing a vessel with Hb or with L-NMMA or by removing the endothelium, from which we conclude that the recovery process requires NO derived from L-arginine by endothelial NOS. The mechanism underlying the s-component is clearly different since it is unaffected by Hb or L-NMMA and so does not depend on endothelium-derived NO, at least not directly. Formally, then, the difference between VIS and UVA light-induced photorelaxations can be explained if we postulate that both light sources can release NO from a photodegradable molecular 'store', but only the more energetic UVA light can activate the process(es) that generate(s) the s-component.
There are grounds to suppose that NO might also mediate the s-component, but from a source (or sources) of NO contained within VSM fibres (e.g. as RSNO, haem-NO, NO2-, or some other ligated form). The idea is that NO released photochemically at the onset of illumination would combine with soluble guanylate cyclase (sGC)-haem and activate the enzyme. Under continuous UV irradiation the newly-formed sGC-haem-NO would itself be subject to photolysis. Photolysis of protein-bound nitrosyl-metalloporphyrins is known to yield very low quantum efficiencies for NO as compared to non-protein-bound nitrosyl-metalloporphyrins (Morlino & Rodgers, 1996). This is because the NO released becomes trapped within the molecular matrix of the protein and is unable to diffuse away from the haem group before re-ligation occurs, a process called geminate recombination (Negrerie et al. 1999). Could this provide an explanation for sustained activation of sGC under continuous UVA illumination? Binding of NO to the haem group in sGC deforms the porphyrin ring and this conformational event results in enzyme activation. Repeated cycles of photolysis followed by rapid re-binding of NO to sGC-haem under continuous UVA irradiation might set up a cyclical deformation of the porphyrin ring, causing the enzyme to alternate between active and inactive states for as long as the vessel remains illuminated.
The possibility that sGC might be activated by light in a truly NO-independent manner should also be considered. Stasch et al. (2001) identified a site on the 
1 subunit of the enzyme that is stimulated by the pyrazalopyridine BAY 41-2272, in a haem-dependent manner. Activation by this means was not affected by NO scavengers but it was prevented by ODQ, a selective inhibitor of sGC. The underlying mechanism is not fully understood, but it is known to involve cysteine residues 238 and 243.
Several characteristics of the s-component are consistent with a direct effect of light on sGC. First, it is maintained throughout the period of illumination and only reverses when the light is switched off. Rodriguez et al. (2003) were able to record protracted s-type responses lasting up to 2 h. Their measurements of the action spectra suggested that photodecomposition of nitrite anions was responsible and this was supported by biochemical assays showing that vessels contained appreciable amounts of nitrite (~10 µM). Second, the failure of either Hb or L-NMMA to prevent the s-component is consistent with the model outlined above, since the NO 'trapped' within the enzyme matrix would not be accessible to exogenous (extracellular) Hb, nor would it be affected by inhibiting NOS with L-NMMA. (Some comment on the inability of Hb to block the s-component is needed here, since this result is at odds with previously published data. A possible explanation is that Hb was delivered to the interior of the vessel in our study, while others have generally added it to the exterior. The latter would result in the absorption of a fraction of the incident light before reaching the preparation. The extent to which this would screen the preparation and attenuate the response would depend upon the [Hb] and the path length). Third, the observation that endothelium-denuded vessels relax when irradiated with UVA light can be explained, since a pre-formed 'internal' source of ligated NO will be present in VSM fibres prior to removal of the endothelium. We can assume that this 'captive' NO is limited in amount in endothelium-denuded vessels and so could become depleted if repeatedly irradiated. Indeed, the literature shows that endothelium-deficient preparations exhibit a stepwise loss of photosensitivity under these conditions (Venturini et al. 1993; Charpie et al. 1994), consistent with a gradual 'leakage' of NO from VSM cells (Kubaszewski et al. 1994). Interestingly, the photosensitivity of preparations with intact endothelium is maintained almost indefinitely (Rodriguez et al. 2003). Finally, the findings that inhibitors of sGC abolish this type of photorelaxation (Martin et al. 1985) and that UV light has been shown to activate crude sGC preparations directly (Karlsson et al. 1984, 1985, 1986) lend further credence to this hypothesis.
Quantitative considerations
Some comment is necessary on two matters that have a direct bearing on our quantitative analyses.
First, we have implied that the amplitude of a photorelaxation is determined solely by the quantity of NO produced photochemically, and that this reflects the overall capacity of the store. However, the biological efficacy (or bioactivity) of the NO released will influence the size of a response and it is essential to take account of this when drawing comparisons between vessels from normotensive versus hypertensive animals, or when comparing VIS with UVA light-induced photorelaxations. The near diffusion rate-limited reaction of NO with the superoxide anion (O2-) to form peroxynitrite (ONOO-; Beckman & Crow, 1993) is especially relevant because O2- production is increased in hypertension (Grunfeld et al. 1995; Tschudi et al. 1996; Bauersachs et al. 1998; Brovkovych et al. 1999). Furthermore, UV light can generate O2- directly by photolysis of water (McCord & Fridovich, 1973).
Second, the relative contributions of p- and s-components to the UVA light-induced response were roughly comparable under the conditions of our experiments. The irradiance obtained with the UVA source used here was less than that of the VIS source (~20 %), and while it was probably enough to fully discharge the molecular store of NO, it may not have been sufficient to achieve maximal activation of the s-component. This point is considered again later.
NO-dependent photorelaxation is impaired in arteries from spontaneously hypertensive rats
Repriming of VIS light-induced photorelaxation is attenuated in vessels from SHR when compared to that seen using arteries from NTR: analysis shows that P/Pmax and T1/2 values are significantly reduced to around 50 % and 40 %, respectively. This result conflicts with several reports in the literature showing that photorelaxation is enhanced in vessels from hypertensive animals (Kubaszewski et al. 1994; Charpie et al. 1994). There are several points that have a bearing on this discrepancy. First, most investigators used UVA light only when comparing arteries from normotensive and hypertensive animals and did not undertake parallel experiments with VIS light. Second, with the exception of the recent study by Rodriguez et al. (2003), the distinction between phasic and sustained elements of the UVA light-induced response has not been made. Finally, in most instances vessels lacking an intact endothelium were studied.
Our experiments with functionally intact vessels from SHR show that the repriming curve for the p-component of UVA light-induced responses is not significantly different from that seen when arteries are illuminated with VIS light. This similarity, highlighted in Fig. 8, shows that the choice of wavelength does not influence repriming of vessels from SHR. The situation is very different when arteries from NTR are studied. Here, the recovery of a vessel exposed to UVA light is greatly impaired when compared to one irradiated with VIS light: P/Pmax and T1/2 are reduced to 24 % and 15 %, respectively. This result suggests that arteries from NTR are susceptible to damage by UVA light while those from SHR are more resistant. The difference could explain why previous authors have concluded that photorelaxation is enhanced in hypertension. The most meaningful comparison to be made is between the repriming curve for vessels from NTR irradiated with VIS light (upper curve, Fig. 6A and the two curves for arteries from SHR shown in Fig. 8). These data show that NO-dependent photorelaxation is compromised in vessels from SHR.
The results of this study suggest that the overall capacity of the photosensitive store is reduced in SHR, due to a deficiency in one (or more) of the reactants required for its synthesis, or that the bioactivity of the NO generated photochemically is impaired. It seems probable that both factors are involved, since defective NO production (Malinski et al. 1993; Brovkovych et al. 1999) as well as enhanced O2- synthesis have each been implicated in the pathogenesis of hypertension (Linder et al. 1990; Panza et al. 1990; Nakazono et al. 1991; Treasure et al. 1992; Taddei et al. 1993; Grunfeld et al. 1995; Tschudi et al. 1996). Furthermore, there is evidence to show that the expression of sGC is down-regulated in hypertension (Bauersachs et al. 1998). Thus, the reduced photosensitivity of vessels from SHR could be attributable to any one of these factors operating independently, or all acting together.
The s-component of the UVA response is enhanced in vessels from spontaneously hypertensive animals
Most authors have studied the s-component in isolation, albeit inadvertently, by using endothelium-denuded vessels irradiated with UV light. Our results show that the s-component is increased approximately 2-fold in vessels from SHR as compared to those from NTR. It is possible that we underestimate the real magnitude of the s-component because much larger responses than those obtained here (~90 % relaxation) have been reported previously under some conditions (Chang et al. 1993; Charpie et al. 1994). Since the p-component of the UVA light-induced response is actually reduced in SHR, reports of enhanced photorelaxations exhibited by vessels from hypertensive animals must actually reflect an increase in the amplitude of the s-component.
Finally, our experiments show that photorelaxation in response to either UVA or VIS light is profoundly affected by the prior history of a vessel, particularly by the duration of the interval between successive periods of irradiation. The tractive force associated with fluid flow, called shear stress, is an important determinant of NO production in vivo, since it stimulates both the synthesis and release of endothelial NO (Kanai et al. 1995). It is possible then that the rate of flow of perfusate through an intact artery could significantly influence repriming of both VIS- and UVA light-induced responses, though we did not investigate this in the present study.
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Acknowledgements
This study was supported by a Project Grant to F. W. F. from the British Heart Foundation (no. PG/93116). We thank Miss Carol Scott for help in performing the experiments and David Roche, Jim Allen and Sean Earnshaw for expert assistance in preparing the figures.
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) of the responses generated by irradiating vessels with UVA light. The method of analysing original recordings is described in the text. Scale bar = 5 %.
; from Fig. 6B).